Involvement of pectin methyl-esterase during the ripening of grape berries: partial cDNA isolation, transcript expression and changes in the degree of methyl-esterification of cell wall pectins.

Grape berries (Vitis vinifera L., cv Ugni blanc) were harvested at 12 different weeks of development in 1996 and 1997. Ripening was induced at veraison, the crucial stage of berry softening, and was followed by a rapid accumulation of glucose and fructose and an increase of pH. Total RNAs, crude proteins and cell wall material were isolated from each developmental stage. A partial length cDNA (pme1, accession number AF159122, GenBank) encoding a pectin methyl-esterase (PME, EC 3.1.1.11) was cloned by RT-PCR with degenerate primers. Northern blots revealed that mRNAs coding for PME accumulate from one week before the onset of ripening until complete maturity, indicating that this transcript represents an early marker of veraison and could be involved in berry softening. However, PME activity was detected during all developmental stages. Total activity per berry increased, whereas "specific" activity, on a fresh weight basis, decreased during development. The amount of cell wall material (per berry and per g of berry) followed the same pattern as that of PME activity (total and "specific" respectively), indicating they were tightly correlated and that PME levels varied very little in the cell walls. Nevertheless, the degree of methyl-esterification of insoluble pectins decreased throughout the development from 68% in green stages to less than 20% for the ripe berries, and this observation is consistent with the induction of PME mRNAs during ripening. Relations between transcript expression, PME activity, the DE of insoluble pectic polysaccharides and their involvement in grape berry ripening are discussed.

Changes in acidity and in proton transport at the tonoplast of grape berries during development.

As in many fruits, the induction of grape berry (Vitis vinifera L.) ripening results in intense breakdown of malic acid. Using membrane fractions, we tested the hypothesis that changes in acidity resulted from malate vacuolar decompartmentation. The hydrolytic activities of the two primary vacuolar pumps inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) and vacuolar ATPase (V-ATPase; EC 3.6.1.3) increased throughout development with an acceleration during ripening, as confirmed by Western blotting and analysis of transcript expression. The ratio of V-PPase activity to V-ATPase activity was always in favour of V-PPase and reached its maximum value at véraison. The rate of anion transport strongly increased during ripening. Before ripening, tonoplast passive permeability was low, but rose during ripening. Our data indicate that tonoplast leakage dramatically increased during ripening. This leakage is probably the prime cause of malate decompartmentation, amplified by the incapacity of oxidative phosphorylation to face increased energy demand.

Cloning and expression of a hexose transporter gene expressed during the ripening of grape berry.

The ripening of grape (Vitis vinifera L.) is characterized by massive sugar import into the berries. The events triggering this process and the pathways of assimilate transport are still poorly known. A genomic clone Vvht1 (Vitis vinifera hexose transporter1) and the corresponding cDNA encoding a hexose transporter whose expression is induced during berry ripening have been isolated. Vvht1 is expressed mainly in the berries, with a first peak of expression at anthesis, and a second peak about 5 weeks after véraison (a viniculture term for the inception of ripening). Vvht is strictly conserved between two grape cultivars (Pinot Noir and Ugni-Blanc). The organization of the Vvht1 genomic sequence is homologous to that of the Arabidopsis hexose transporter, but differs strongly from that of the Chlorella kessleri hexose transporter genes. The Vvht1 promoter sequence contains several potential regulating cis elements, including ethylene-, abscisic acid-, and sugar-responsive boxes. Comparison of the Vvht1 promoter with the promoter of grape alcohol dehydrogenase, which is expressed at the same time during ripening, also allowed the identification of a 15-bp consensus sequence, which suggests a possible co-regulation of the expression of these genes. The expression of Vvht1 during ripening indicates that sucrose is at least partially cleaved before uptake into the flesh cells.

Subunit E of the vacuolar H(+)-ATPase of Hordeum vulgare L.: cDNA cloning, expression and immunological analysis.

A tonoplast protein of 31 kDa apparent molecular mass (TpP 31) was isolated from two-dimensional gels. Amino acid sequences were determined from LysC endoproteinase-peptide fragments. Using degenerate oligonucleotides, a corresponding cDNA clone of 1034 bp was isolated from a barley leaf cDNA library. It encodes for subunit E of the vacuolar H(+)-ATPase, the first one identified in plants so far. The open reading frame extends over 681 bp, encoding a gene product of 227 amino acids and a calculated molecular weight of 26,228 g mol-1. Northern and Western blot analysis indicates constitutive expression of subunit E in all plant organs with only small effects of salt stress. Localization of TpP 31 at the tonoplast was confirmed in fractions of purified vacuolar membrane obtained by free-flow electrophoresis. Immunoprecipitation of newly synthesized 35S-labelled membrane proteins with anti-TpP 31 gave two additional bands with apparent molecular masses of about 53 and 62 kDa. Gel filtration after mild solubilization showed co-purification of TpP 31 with the 55 kDa subunit of the H(+)-ATPase. Both results provide evidence beyond the sequence homology that TpP 31 is a structural component of the vacuolar H(+)-ATPase.

Potassium stimulation of corn root plasmalemma ATPase : I. Hydrolytic activity of native vesicles and purified enzyme.

Potassium stimulation of the plasmalemma (Zea mays L. var Mona) was studied by using a constant ionic strength to prevent indirect stimulation by the electrostatic effect of K(+) salts. The transmembrane electrochemical H(+) gradient was eliminated by using gramicidin. In these conditions, K(+) stimulation was attributable to a direct effect of the cation on plasmalemma proteins. We used both native vesicles isolated on a sucrose cushion, and solubilized and purified ATPase from phase-partitioned plasmalemma, according to the method of T. Nagao, W. Sasakawa, and T. Sugiyama ([1987] Plant Cell Physiol 28: 1181-1186). The purified enzyme had a high specific activity (15 micromoles per minute per milligram protein), but was only about 20% stimulated by K(+). In both preparations, potassium (in the range around 1 millimolar) specifically decreased two-fold the vanadate inhibition constant, and increased the maximum rate of ATP hydrolysis. In plasmalemma vesicles, the Eadie-Scatchard graph of the K(+)-dependent ATPase activity as a function of K(+) concentration was linear only at constant ionic strength. The purified ATPase preparation appeared as two closely spaced bands in the 100 kilodalton region with isoelectric point about 6.5. Nevertheless, this biochemical heterogeneity seems unlikely to be related to K(+) stimulation, since K(+) modified neither the pH optimum of the activity (pH 6.5) nor the monophasic kinetics of the vanadate inhibition, in both native plasmalemma and purified enzyme preparation.